New substrates for diagnostic use, with high susceptibility to trypsin and other proteolytic enzymes of the type peptide peptidohydrolases

ABSTRACT

Substrate with high susceptibility to peptide peptidohydrolases represented by the formula:   OR ITS SALTS, WHERE R1 is hydrogen, an alkyl-carbonyl having 112 carbon atoms, a omega -aminoalkyl-carbonyl having 1-12 carbon atoms in a straight chain, cyclohexyl-carbonyl, a omega cyclohexylalkyl-carbonyl having 1-6 carbon atoms in a straight chain, 4-aminomethyl-cyclohexyl-carbonyl, benzoyl, a omega phenylalkylcarbonyl having 1-6 carbon atoms in a straight chain, benzene-sulphonyl or 4-toluene-sulphonyl; R2 is hydrogen, phenyl, cyclohexyl, or an alkyl having 1-6 carbon atoms; X is methylene or a single bond; R3 is a straight, branched or cyclic alkyl group having 3-8 carbon atoms; R4 is a straight, branched or cyclic alkyl group having 3-8 carbon atoms, phenyl or benzyl; n is 3 or 4; R5 is hydrogen or guanyl; and R6 is phenyl, nitrophenyl, methylnitrophenyl, dinitrophenyl, naphthyl, nitronaphthyl, quinolyl, or nitroquinolyl.

United States Patent Claeson et a1.

[11] 3,886,136 51 May 27, 1975 PEPTIDOHYDROLASES Inventors: Karl GoranClaeson; Birgitta Gunilla Karlsson, both of Goteborg; Lars-GundroSvendsen, Molndal, all of Sweden Assignee: AB Bofors, Bofors, SwedenFiled: Apr. 24, 1973 Appl. No.: 354,038

[30] Foreign Application Priority Data May 2, 1972 Sweden 5758/72 US.Cl. 260/1125; 195/103.5; 424/177 Int. CL. C07c 103/52; C07g 7/00; A6lk27/00 Field of Search 260/1 12.5

References Cited OTHER PUBLICATIONS Nachlas et al., Arch. Biochem.Biophys., 108, 266-274 (1964). Plapinger et al., J. Org. Chem., 301781-1785 (1965).

[57] ABSTRACT Substrate with high susceptibility to peptidepeptidohydrolases represented by the formula:

R N X CH C NH CH or its salts, where R is hydrogen, an alkyl-carbonylhaving 1-l2 carbon atoms, a w-aminoalkyl-carbonyl having 112 carbonatoms: in a straight chain, cyclohexyl-carbonyl, am-cyclohexylalkyl-carbonyl having 16 carbon atoms in a straight chain,4- aminomethyl-cyclohexyl-carbonyl, benzoyl, a m-phenylalkylcarbonylhaving l-6 carbon atoms in a straight chain, benzene-sulphonyl or4-to1uenesulphonyl; R is hydrogen, phenyl, cyclohexyl, or an alkylhaving l-6 carbon atoms; X is methylene or a single bond; R is astraight, branched or cyclic alkyl group having 3 8 carbon atoms; R, isa straight, branched or cyclic alkyl group having 3-8 carbon atoms,phenylor benzyl; n is 3 or 4; R is hydrogen or guanyl; and R is phenyl,nitrophenyl, methylnitrophenyl, dinitrophenyl, naphthyl, nitronaphthyl,quinolyl, or nitroquinolyl.

v "19 Claims, N0 Drawings HIGH SUSCEPTIBILITY TO TRYPSIN AND OTHERPROTEOLYTIC ENZYMES OF THE TYPE PEPTIDE PEPTIDOHYDROLASES The presentinvention relates to new substrates for diagnostic use with highsusceptibility to proteolytic enzymes of the type peptidepeptidohydrolases. The substrates according to the invention areintended for quantitative determination of classified and hithertounclassified enzymes of the type E.C. 3.4.4., especially such that breakdown peptides or proteins in the peptide chain at the carboxylic side ofarginine or lysine; e. g., trypsin, thrombin, plasmin, Reptilase fromPentapharm, Basel, Switzerland, Arvine from Ferring AB, Malmo, Sweden,and the hitherto unclassified enzyme Brinase from AB Astra, Sodertalje,Sweden. The substrates may further be used for the study of reactions inwhich such enzymes are formed, inhibited or consumed, and also for thedetermination of factors which affect or participate in such reactions,for example the determination of proenzymes, activators, antienzymes andenzyme inhibitors.

The Enzyme Nomenclature recommended by The International Union ofBiochemistry Elsevier, Amsterdam, 1965 was used in the classification ofthe enzymes.

Compounds (substrates) which have previously been used for quantitativedetermination of the above mentioned enzymes are described in Methodender enzymatischen Analyse, Vol. I, p. 1023 (Ed. Bergmeyer, H. U., VerlagChemie, l970). Depending upon which of the catalytic reactions of theproteolytic enzymes a. chromophoric products that are easy to measurespectrophotometrically and have light absorption maxima which do notcoincide with those of the original amide substrates;

b. fluorescent products which may be measured by means of fluorescencespectrophotometry;

c. products which, after coupling with a suitable reagent give rise tocoupling products which may be photometrically measured with highsensitivity.

Some synthetic amide substrates with hydrolysable chromophoric groupshave come into use. These are primarily of the types N -unsubstitutedand N substituted mono-aminoacid-p-nitroanilide derivatives and monoamino acid-B-ruaphtylamide derivatives.-

Among these substances N -benzoyl-DL-arginine-pnitroanilinehydrochloride (BAPNA) may be mentioned as a reagent for trypsin (E.C.3.4.4.) and for re actions in which trypsin participates. The enzymatichydrolysis of this substrate produces the chromophoric productp-nitroaniline, which may readily be measured spectrophotometrically.

However, these earlier known amide substrates do not possess the desiredspecificity and sensitivity. This is a significant draw-back, which maygive rise to a more involved procedure in the taking of specimen,because a considerable amount of biological material will then berequired. Furthermore, the enzymatic reaction time is long and theprecision of the enzyme determination may be unsatisfactory.

The new substrates of the amide type according to the invention whichhave a very high susceptibility to peptide peptidohydrolases arerepresented by the following general formula:

I R -N-X-CH-iE-NH--("JH-i:-NH-CH- -NH-R R R R IIIH 5 that takes placethe esterolytic or the amidolytic or its salts, where R, may be selectedfrom hydrogen,

these synthetic substrates may in principle be divided into two maingroups: ester substrates and amide substrates. The largest group ofsynthetic substrates as previously used is the group of estersubstrates. This depends on the fact that these are converted much morerapidly by the peptide peptidohydrolases than the amide substrateshitherto produced. However, the principal biological function of theenzymes classified as peptide peptidohydrolases is, as evident from thename; to hydrolyze peptide or amide bonds, but not ester bonds, ofnatural substrates. In the literature (Blood Clotting Enzymology, p. 36and 42 44, Ed.: Seegers -W. H., Academie Press, 1967) it is reportedthat the ratio between the reaction velocities of the esterolytic andthe amidolytic catalyses of thrombin is not constant under differentreaction conditions. For this reason, synthetic amide substrates whichhave much greater susceptibility to the enzymes in question, and whichalso more rapidly may be broken down to measurable products that thosehitherto known, have been desirable.

In order to study and follow up the reaction course of the enzymatichydrolysis the amide substrates are particularly suitable since they maygive:

an acyl having from 1 to 12 carbon atoms, a w-aminoacyl having from 1 to12 carbon atoms, cyclohexylcarbonyl, a w-cyclohexyl-acyl,4-aminomethyl-cyclohexylcarbonyl, benzoyl, a benzoyl substituted withe.g., one or more halogen atoms, methyl-, aminoor phenylgroups, etc., aw-phenyl-acyl having from 1 to 6 carbon atoms in the acyl part and wherethe phenyl group may be substituted, benzenesulphonyl and4-toluenesulphonyl.

R may be selected from hydrogen, phenyl and an alkyl having from 1 6carbon atoms and cyclohexyl.

X may be selected from methyleneand a single bond.

R may be selected from a straight, branched or cyclic alkyl group havingfrom 3 to 8 carbon atoms.

R, may be selected from a straight, branched or cyclic alkyl grouphaving from 3-8 carbon atoms, phenyl and benyzl.

n may be selected from 2, 3 and 4.

R may be selected from hydrogen and guanyl.

R may be selected from phenyl, nitrophenyl, methylnitrophenyl,dinitrophenyl, naphthyl, nitronaphthyl, quinolyl and nitroquinolyl.

The new substrates may be produced according to two principallydifferent methods.

1. The first method is based upon the coupling of the chromophoric groupR to the amino acid in question, and then a step-by-step building up ofthe desired peptide structure by means of gradual coupling of theremaining amino acids. The chromophoric groupis here used as a blockinggroup for the C-terminal carboxyl group of the first amino acid.

2. The other method is based upon a step-by-step building up of thedesired peptide structure and upon the'subsequent removal of usedblocking groups, and finally coupling of the chromophoric group R to thepeptide structure.

In the stepwise synthesis of the peptide derivatives suchcouplingmethods have been used that are well known and-commonly used inthe peptide chemistry. Such well known blocking groups, that arecommonly used within thepeptide chemistry, as for example Cbo(carbobenzoxy), MeOCbo (p-methoxycarbobenzoxy)- NO Cbo(p-nitrocarbobenzoxy), MCbo (p-methoxyphenylazo-carbobenzoxy), BOC(tertbutyloxycarbonyl), TFA (trifluoroacetyl) or formyl are used asamino blocking groups. The oz-carboxyl group can 'beactivated by meansof conversion to different activated, in peptide chemistry well knownand often used'derivatives, which may either be isolated or be generatedin situ, as for example p-nitrophenylester, trichlorophenylester,pentachlorophenylester, N-hydroxysuccinimideester, acid azide, acidanhydride, which may either be symmetric or unsymmetric. It may also beactivated with a carbodiimide such as N,- N-dicyclohexylcarbodiimide.The C-terminal carboxyl group in the amino peptide derivative or theamino acid derivative may be protected by esterifying to e.g., m ethyl-,ethylor isopropylester or by means of conversion to the chromophoricaniline derivative, which thus works as a blocking group during thebuilding up of the peptide chain. Those free functional groups which donot take part in the reaction, may during the synthesis of the peptidesor the peptide derivatives be protected in the following manner:

For the purpose of blocking the arginyl S-guanido group and the lysyle-amino group, one may use such amino blocking groups, commonly usedwithin peptide chemistry, as for example N0 Tos (ptoluenesulphonyl) oronly protonization as protection for the guanido group, and Cbo(carbobenzoxy), BOC (tert. butyloxycarbonyl) or also Tos for the e-aminogroup. As protection for the hydroxyl group in tyrosin one may use suchblocking groups, commonly used within the peptide chemistry, as forexample benzyl and tertiary butyl protection groups.

In the stepwise synthesis of the peptide structure a systematicpurification by means of gel filtration may be carried out after eachcoupling of a new amino acid. For this gel filtration a column is usedwhich is packed with a material suitable for the gel filtration, e.g., across-linked dextran gel of the type Sephadex G or LH from PharmaciaFine Chemicals, Uppsala, Sweden. Another suitable gel consists ofcopolymers of vinylacetate e.g. of the type Merckogel OR-PVA from A G E.Merck, Darmstadt, West-Germany. The gel material is used equilibratedwith a suitable solvent and elution is then carried out with the samesolvent, e.g., methanol, ethanol, acetone, dimethylformamide,

etamide, dimethylsulphoxide or hexamethylphosphoric triamide. k

The invention will be described more in detail in the following exampleswhich demonstrate the production of different substrates according tothe invention by means of stepwise synthesis. However, these examples donot limit the scope of invention.

In the thin layer chromatographic analysis of the eluate and theproducts glass plates were used with silica gel (F 254 from A G E.Merck, Darmstadt, West- Germany) as absorption medium. For thedevelopment of the thin layer chromatograms the following solventsystems have been used:

A: n-butanolzacetic acidzwater (3:121) C: n-Eropanokethyl acetatezwater(7:1:2) D: neptanezn-butanolzacetic acid (312:1) P chloroformcmethanol(9:1

After the thin layer chromatographing, the plates were developed firstin UV light (254 nm), and subsequently with the chlorine/toluidinereaction (Ref.: G. Pataki: Dunnschluchtchromatografie in der Aminosaureund Peptid-Chemie, Walter de Gruyter & Co., Berlin, 1966, p. as adevelopment method.

Unless otherwise stated all amino acids used have the L-configuration,and the abbreviations have the following meanings:

Ala Alanine Arg Arginine lle lsoleucine OisoPr q'so-Propyloxy pNA=p-Nitroanilide Tos arToluenesulphonyl TLC =Thin layer chromatographyThe gels Sephadex G-l5 and G-25 as used for the gel filtration are bothcross-linked dextran gels with different cross-linking degree fromPharmacia Fine Chemicals, Uppsala, Sweden. The gel Sephadex w LM-20 is ahydroxypropylated crosslinked dextran gel from Pharmacia Fine Chemicals,Uppsala, Sweden.

EXAMPLE I H-Leu-Leu-Arg-pNA-ZHCl Example la: Cbo- Arg (N0 pNA 35,3 g(0.1 mole) of dry Cbo Arg (N0 OH are dissolved in 200 ml f freshlydestilled HMPTA at room-temperature, where upon 10.1 g (0.1 mole) of EtN and 24.6 g (0.15 mole) of p-nitrophenylisocyanate are added withstirring under completely dry conditions. After 24 hours atroomtemperature the reaction mixture is poured into 2 l. of

r a 2% sodium-bicarbonate solution while stirring. The

Yield: 29.8 g (63.0%) of la, m.p. 185-188C, homogeneous according to TLCin P, and C, and ML, 1.3 (c =1.l;-AcOH).

Example lb: Cbo Leu Arg (N pNA Method of synthesis: 5.0 g (10.6 mmoles)of la are dissolved in 21 ml of AcOH and 22 ml of 4 N HBr in AcOH undercompletely dry conditions. The reaction mixture is stirred for one hour,and thereafter it is slowly poured into 200 ml of vigerously stirredether, and 1.5 HBr H Arg (N0 pNA precipitates out. The ether solution isdecanted and the granular residue is treated another three times with100 ml of ether in order to remove benzyl bromide and the surplus of HBrand AcOH. After drying in vacuum over P 0 the yield of the hydrobromidesalt of the amino acid derivate is quantitative (4.87 g). 4.87 g(10.6.mmoles) of 1.5 HBr H -.Arg (N0 pNA are dissolved in 50 ml ofdestilled DMF. The solution is cooled to l0C and 1.6 g

(15.9 mmoles) of Et N are added to liberate H Arg (N0 pNA from itshydrobromide salt. The mixture is allowed to react for 1 hour under dryconditions. Precipitated Et N.HBr is filtered off and the filtrate iscooled to C. 4.3 g (12.7 mmoles) of Cbo Leu OpNP are added and thesolution may now slowly arise to room temperature. After 3 hours thesolution is cooled again to l0C and buffered with 0.55 g (5 mmoles) ofEt N. The buffering procedure is repeated once more after 2-3 hours.After 24 hours of reaction time the solution is evaporated at 40C invacuum to dryness. The residue is treated three times with 100 ml ofdestilled water and it is thereafter dried in vacuum. Purification: Thedry residue is dissolved in MeOH and purified by gel chromatography on acolumn of Sephadex LH- equilibrated with MeOH. Yield: 6.1 g (98%) ofamorphous lb, homogeneous according to TLC in P and [(11 3 33.3 (c 1.0;MeOH).

Example lc: Cbo Leu Leu Arg (N0 pNA Starting materials: 2.8 g (4.77mmoles) of lb and 2.22 g (5.72 mmoles) of Cbo Leu OpNP.

Method of synthesis: According to Example lb.

Purification: Gel chromatography on Sephadex LH-2O in MeOH. Yield 2.5 g(80%) of amorphous 1c, homogeneous according to TLC in P and [01 =9.9 (c1.0; DMF).

Example Id H Leu Leu Arg pNA 2HC1 134,2 mg (0.192 mmole) of 1c areplaced in the reaction vessel of a- Sakakibara apparatus. 5 ml of dryhydrogen fluoride are destilled over into the vessel and allowed toreact for one hour under stirring. Thus the nitro group protecting theguanidino function of arginine and the Cbo-group are cleaved off.Afterwards the hydrogen fluoride is destilled off under reduced pressureand the dry residue is dissolved in DMF. In order to convert theobtained hydrofluoridederivate of the peptide to its hydrochloride salt,0.25 ml of concentrated hydrochloric acid is added to the DMF-solution.After evaporation the procedure-of'conversion is repeated once more. 1 I

Purification: The residue after the last evaporation is dissolved in asolution of 50% AcOH and is purified by gelchromatography on a column ofSephadexG-25 equilibrated with 50% AcOH. The fraction of theeluatecontaining the pure tripeptide hydrochloride derivate was lyophilized.Yield: 80.0 mg (71%) of amorphous I, chlorine content 11.83%,homogeneous according to TLC in A and [011 =29.9 (c 0.59; 5 0% AcOH):Amino acid analysis showed the following proportions: Leu: 2.0; Arg:0.95.

Example 11; N Bz Leu Leu Arg -pNA HCl 11 Example 11a: N Bz Leu Leu Arg(N02) pNA Starting materials: 703 mg (1 mmole) of 1c and 272 mg (1.2mmoles) of B2 0.

Method of synthesis: Decarbobenzoxylation of 1c is carried out accordingto Example 1b. The dry product ofH Leu Leu Arg (N0 pNA HBr is filteredoff. The filtrate is cooled to 10C and 272 mg (1.2 mmoles) of B2 0 areadded. After 3 hours of reaction time the solution has reached roomtemperature. lt is cooled again and buffered with 0.07 ml (0.5 mmole) ofEt N. The procedure of buffering is repeated after another 3 hours.After 24 hours of reaction time the solution is evaporated in vacuum todryness. The residue is treated and dried according to the proceduredescribed in Example Ib.

Purification: Gel chromatography on Sephadex LH-20 in MeOH.

Yield: 550 mg (82%) of amorphous 11a, homogeneous according to TLC in Pand [041 3.6 (c 1.01; DMF).

11: N B2 Leu Leu Arg -pNA HCl Starting material: 554.8 mg (0.828 mmole)of 11a.

Method of synthesis: According to Example 1d.

Purification: Gel chromatography on Sephadex G-15 in 20% AcOH andSephadex LH-20 in MeOH. Yield: 476 mg (87%) of lyophilized, amorphousI1, chlorine content 5.30 homogeneous according to TLC in A and [0:],{73,0 (c 0.66; 50% AcOH). Amino acid analysis showed the followingproportions: Leu: 2.0, Arg: 0.95.

Example lIl: H B cyclohexyl Ala Val -Arg -pNA 2 HCl 111a. Cbo Val Arg(N0 pNA Starting materials: 20.6 g (43.5 mmoles) of la and 20.3 g (54.3mmoles) of Cbo Val OpNP.

Method of synthesis: According to Example'lb.

Purification: Recrystallization of the crude product from MeOH. Themother liquor is purified by gel chromatography on Sephadex LH-20, inMeOH. Yield: 23.2 g (93.3%) of 111a, nip. 200-202C, homogeneousaccording to TLC inP and C and [01],, +5.8 (c 1.0; DMF).

lllb. Cbo B cyclohexyl Ala Val Arg (N0 pNA Starting materials: 0.48 g(0.83 mmole) of Illa and 0.53 g (1.24 mmoles) of Cbo B cyclohexyl AlaOpNP having a m.p. of ll06C and [a] =28.8 (c 1.0; DMF).

Method of synthesis: According to Example lb.

Purification: Gel chromatography-on Sephadex LI-l-20 is MeOH.

Yield: 531 mg (88%) of amorphous lllb, homogeneous according to TLC in Pand [01] 7.3 (c 2.0; DMF).

111. H B cyclohexyl Ala Val Arg pNA 2 HC] Starting material: 120.4 mg(0.165 mmole) of 111 b.

Method of synthesis: According to Example Id.

Purification: Gel chromatography on Sephadex G-l5 in 20% AcOl-l. Yield:56.6 mg (55%) of lyophilized, amorphous Ill, chlorine content 11.32%,homogeneous according to TLC in A, [041 36.8 (c 0.62; 50% AcOH). Aminoacid analysis showed the folowing proportions: Val: 1.0, B-cyklohexylAla 1.1; Arg: 1.0.

Example IV: N Bz B cyclohexyl Ala Val Arg pNA HCl IVa. N Bz B cyclohexylAla Val Arg (N0 pNA Starting materials: 243 mg (0.335 mmole) of Illb and93 mg (0.41 mmole) of B2 0.

Method of synthesis: According to Example Ila.

Purification: Gel chromatography on Sephadex LII-20 in MeOH. Yield: 147mg (63%) of IVa, m.p. l48152C, homogeneous according to TLC in P and[01],, 6.33 (c 0.84; DMF).

IV. N Bz B cyclohexyl Ala Val Arg pNA HCl Starting material: 106.0 mg(0.152 mmole) of lVa.

Method of synthesis: According to Example Id.

Purification: Gel chromatography on Sephadex G- in AcOI-I. Yield: 86.7mg (84%) of lyophilized, amorphous IV, chlorine content 5.10%,homogeneous according to TLC in A and [011 -77 (c 0.3; 50% AcOH). Aminoacid analysis showed the following proportions: Val 1.0, B-cyklohexylAla 1.2, Arg: 1.0.

Example V: N-Bz-Ncyclohexyl-B-Ala-Val-Arg-pNA.HCl

Va. N-Cbo-N-cyclohexyl-B-Ala-Val-Arg(NO )-pNA 8 Yield: 243.5 mg (87%) ofamorphous V b, homogeneous according to TLC in P, and [01],, 05 (C 0.43;DMF.)

N-Bz-N-cyclohexyl-B-Ala-Val-ArgpNA.HCl

Starting material: 170 mg (0.242 mmole) of V b. Method of synthesis:According to Example 1d. Purification: Gel chromatography on SephadexLH-20 in MeOH.

Yield: 126 mg (76%) of lyophilized, amorphous V, chlorine content 5.11%,homogeneous according to TLC in A, [01],, 38.5 (C 0.69; 50% AcOl-l).Amino acid analysis showed the following proportions: Val: 1.0,N-cyclohexyl-B-Ala: 1.3, Arg: 0 .9.

Example VI: N -Bz-Val-Arg-'pNA.HCl

Vla. Cbo-Val-Val-Arg(NO )-pNA Starting materials: 1.97 g (3.43 mmoles)of Illa and 1.6 g (4.2 m moles) of Cbo-Val-OpNP.

Method of synthesis: According to Example I b.

Purification: Gel chromatography on Sephadex LII-20 in MeOH. Yield: 1.9g (82%) of amorphous Vla, homogeneous according to TLC in P and C.

Vlb. N -Bz-Val-Val-Arg(NO )-pNA Starting materials: 1.9 g (2.83 mmoles)of VI 0 and 0.77 g(3.40 mmoles) of B2 0.

Method of synthesis: According to Example Ila.

Purification: Gel chromatography on Sephadex LII-20 in MeOH. Yield: 1.45g of amorphous Vlb, homogeneous according to TLC in P and [01],, 5.6 (C1.01; DMF).

VI. N -Bz-Val-Val-Arg-pNA.l-IC1 Starting material: 362 mg (0.564 mmole)of Vlb.

Method of synthesis: According to Example 1d.

Purification: Gel chromatography on Sephadex G-15 in 33% AcOl-l. Yield:248 mg(69.7%) of lyophilized amorphous Vl, chlorine content 5.54%,homogeneous according to TLC in A and [11],, -57.0 (C=0.65; 50% AcoI-l).Amino acid analysis showed the following proportions: Val: 2.0, Arg:0.9.

Example Vll: N -Bz-Leu-Val-Arg-pNAllCl.

Vlla. Cbo-Leu-Val-Arg(NO )-pNA Starting material: 1.97 g 3.43 mmoles) ofIlla and 1.7 g (4.2 mmoles) of Cbo-Leu-OpNP.

Method of synthesis: According to Example 1b.

Purification: .Gel chromatography on Sephadex LH-20 in MeOH. Yield: 2.05g (87%) of amorphous Vlla, homogeneous according to TLC in P and C.

Vllb. N" -Bz-Leu-Val-Arg(NO )-pNA Starting materials: 1.75 g (2.55mmoles) of Vlla and 695 mg (3.06 mmoles) of B2 0.

Method of synthesis: According to Example lla.

Purification: Gel chromatography on Sephadex LH-20 in MeOH. Yield: 1.49g (91%) of amorphous Vllb, homogeneous according to TLC in P and [01],,+2.4 (C=l.0l; DMF).

VIII N" -Bz-Leu-Val-Arg-pNA.I-IC1 Example VIII: N-Bz-Ile-Val-Arg-pNA.HCl

VIIa. Cbo-Ile-Val-Arg(NO )-pNA Starting materials: 1.97 g (3.43 mmoles)of 111a and 1.7 g (4.2 mmoles) of CboJle-OpNP.

Method of synthesis: According to Example Ib.

Purification: Gel chromatography on Sephadex LI-I-20 in MeOH. Yield: 2.0g (85%) of amorphous Vllla, homogeneous according to TLC in P Vlllb. N-Bz-Ile-Val-Arg(N02)-PNA Starting materials: 1.75 g (2.55 mmoles) ofVIIIa and 695 mg (3.06 mmoles) of B2 0. Method of synthesis: Accordingto Example Ila.

Purification: Gel chromatography on Sephadex LI-I-20 in MeOH. Yield:1.46 g (89%) of amorphous VIIIb, homogeneous according to TLC in P VIII.N -Bz-Ile-Val-Arg-pNA.I-IC1 Starting material: 319 mg (0.486 mmole) ofVIIIb. Method of synthesis: According to Example Id.

Purification: Gel chromatography on Sephadex G- in 33% AcOI-I. Yield:264 mg (84%) of lyophilized, amorphous VIII, chlorine content 5.43%,homogeneous according to TLC in A and [11],, -29.9 (C=0.59; 50% AcOI-I).Aminoacid analysis showed the following proportions: Val: 1.0; lle: 0.9;Arg: 1.1 I

Example IX. N -Bz-Val-IIe-Arg-pNA.HCl

IXa. Cbo-Ile-Arg(NO )-pNA Starting materials: 4.9 g (10.4 mmoles) of laand 6.2 g (16 mmoles) of Cbo-Ile-OpNP.

Method of synthesis: According to Example lb.

Purification: Gel chromatography on Sephadex LH-2O in MeOH. Yield: 4.75g (78.1%) of partly chrystalline IXa, homogeneous according to TLC in Pand [11],, 23 (c'=1.0; DMF).

IXb. Cbo-Val-I1e-Arg(NOz)-PNA Starting materials: 800 mg (1.37 mmoles)of IX a and 615 mg (1.65 mmoles) of Cbo-Val-OpNP.

Method of synthesis: According to Example Ib.

Purification: Gel chromatography on Sephadex LH- in MeOH. Yield: 833 mg(88.5%) of amorphous IXb, homogeneous according to TLC in P and [01],,3.23 (c=1.1; DMF).

IXc. N -Bz-.Val-Ile-Arg( NO )-pNA Starting materials: 500 mg (0.73mmole) of IXb and 198 mg (0.876 mmole) of B2 0.

10 Method of synthesis: According to Example Ila. Purification: Gelchromatography on Sephadex LII-20 in MeOH. Yield: 374 mg (78%) ofamorphous IXc, homogenous according to TLC in P, and [04],, 1.9"(c=0.99; DMF).

N -Bz-Val-Ile-Arg-pNA.HCI 1x) Starting material: 200 mg (0.305 mmole) ofIXc.

Method of synthesis: According to Example Id.

Purification: Gel chromatography on Sephadex G-15 in 33% AcOH. Yield:153 mg (77.5%) of lyophilized, amorphous IX, chlorine content 5.40%,homogeneous according to TLC in A and [ash =56.4 (c=0.64; 50% A0011).Amino acid analysis showed the following proportions: Val: 1.0; He:1.1;Arg 1.1. t 1 1 Example X: N Val-Leu-Arg-pNALI-ICI Xa.Cbo-Val-Leu-Arg('NO )pNA Starting materials: 880 mg (1.5 mmoles) of 1band 670 mg (1.8 mmoles) of Cbo-Val-OpNP; Method of synthesis: Accordingto Example Ib. Purification: Gel chromatography on Sephadex LII-20 inMeOH. Yield: 882 mg (86%) of amorphous Xa, homogeneous according to TLCin P and [011 6.6 (c=-1.01;

DMF).

Xb. N -Bz-Val-Leu-Arg(NO )-pNA Starting materials: 400 mg (0.583 mmole)of Xa and 160 mg (0.707 mmole) of B2 0.

Method of synthesis: According to Example IIa.

Purification: Gel chromatography on Sephadex LI-I-20 in MeOH. Yield: 279mg (73%) of amorphous Xb, homogeneous according to TLC in P and. [01],,0.4 (c=l.04; DMF).

X. N -Bz-Val-Leu-Arg-pNA.HCl

Starting material: mg (0.23 mmole) of Xb.

Method of synthesis: According to Example I.

Purification: Gel chromatography on Sephadex G-l5 in 33% AcOH. Yield:126 mg (84.5%) of lyophilized, amorphous X, chlorine content 5.45%,homogeneous according to TLC in A and [a 23 5410" (c=0.64; 50% AcOI-I).Amino acid analysis showed the following proportions: Val: 1.0; Leu:1.1; Arg: 1.0.

Example XI: N -Bz-I]e-Ile-Arg-pNA.I-ICI IXa. Cbo-Ile-Ile-Arg(NO )-pNAStarting materials: 800 mg (1.37 mmoles) of IXa and 640 mg (1.66 mmoles)of Cbo-Ile-OpNP.

Methods of synthesis: According to Example lb. Purification: Gelchromatography on Sephadex LH-2O in MeOH. I Yield: 838 mg (87.5%) ofpartly chrystalline XXa, homogeneous according to TLC in P and [(111)5.7 (c= 1.04; DMF).

Starting materials: 440 mg (0.63 mmole) of XIa and 171 mg (0.76 mmole)of B2 0.

Method of synthesis: According to Example Ila.

1 1 Purification: Gel chromatography on Sephadex LH-20 in MeOH. Yield:409 mg (97%) of partly chrystalline Xlb, homogeneous according to TLC inP,.

XI. N -Bz-lle-lle-Arg-pNA.HCl

Starting material: 201 mg (0.3 mmole) of Xlb.

Method of synthesis: According to Example I.

Purification: Gel chromatography on Sephadex G-15 in 33% AcOH. Yield:172 mg (87%) of lyophilized, amorphous X1, chlorine content 5.33%,homogeneous according to TLC in A and [111 57.0. (c=0.67; 50% AcOl-l).Amino acid analysis showed the following proportions: lle: 2.0; Arg:0.9.

Example X11: N Bz Leu Ile Arg pNA l-lCl Xlla. Cbo Leu lle Arg (N pNAStarting materials: 800 mg (1.37 mmoles) of lXa and 640 mg (1.66 mmoles)of Cbo Leu OpNP.

Method of synthesis: According to Example lb. Purification: Gelchromatography on Sephadex Lil-20 in MeOH. 1 Yield: 772 mg (81%) ofamorphous Xlla, homogeneous according to TLC in P and [M 7.3 (c 1.02;DMF).

Xllb. N Bz Leu lle Arg (NO pNA I xn. N Bz Leu 11 Arg pNA I-lCl Startingmaterial: 193 mg (0.288 mmole) of Xllb. Method of synthesis: Accordingto Example 1. Purification: Gel chromatography on Sephadex G-15 in 33%AcOH.

Yield: 166 mg (86%) of lyophilized, amorphous X11, chlorine content5.34%, homogeneous according to TLC in A and [011 51.4 (c 0.67; 50%AcOl-l). Amino acid analysis showed the following proportions: Leu: 1.0;He: 1.2; Arg: 0.9.

Example Xlll: N Bz lle Leu Arg pNA l-lCl XIlla. Cbo Ileu Leu Arg (N0 pNAStarting materials: 880 mg (1.5 mmoles) of lb and 696 mg (1.8 mmoles) ofCbo lleu OpNP.

Method of synthesis: According to Example lb.

Purification: Gel chromatography on Sephadex Ll-lin MeOH. Yield: 750 mg(71%) of amorphous Xllla, homogeneous according to TLC in P, and [01],,9.85 (c 1.05; DMF).

Xlllb. N Bz lleu Leu Arg (N0 pNA Starting materials: 498 mg (0.71 mmole)of Xllla and 193 mg (0.85 mmole) of B2 0.

Method of synthesis: According to Example lla.

Purification: Gel chromatography on Sephadex LH-20 in MeOH.

Yield: 345 mg (73%) of partly chrystalline Xlllb. homogeneous accordingto TLC in P, and [111 5.7C (c 1.04; DMF).

X111. N Bz lle Leu Arg pNA H. Cl

Starting materials: mg (0.254 mmole) of Xlllb.

Method of synthesis: According to Example 1.

Purification: Gel chromatography on Sephadex| G-15 in 33% AcOI-l. Yield:129 mg (77%) of lyophilized, amorphous X111, chlorine content 5.31%,homogeneous according to TLC in A and [041 =49.9 (c 0.68; 50% AcOl-l).Amino acid analysis showed the following proportions: Leu: 1.0; lle:1.1; Arg: 1.05.

Example XIV: N Bz L Leu Leu- Arg -'2; A H01 XlVa. Cbo Arg (N02) 2 NA. vI

3.6 g (10 mmo1es)'of dry Cbo Arg (N02) OH are dissolved in 200 ml ofTHF. 1.0 g (10 mmoles) of Et N are added, where upon the solution iscooled to 10C under completely moisture free conditions.

1.3 g (10 mmoles) of isobutyl chloroformate dissolved in 10 ml of THFare added to the cooled solution during 10 min., and after another 10min. 1.72 g (10 mmoles) of Z-naphthylamine are added. The reactionmixture is allowed to reach room temperature and is left at thistemperature for 24 hours. The reaction mixture is evaporated in vacuumto dryness, is treated 35 times with destilled water, 35 times with a 5%sodium bicarbonate solution and again 3-5 times with destilled water,after which it is dried in vacuum.

Purification: Gel chromatography on Sephadex Ll-l-20 in MeOH; g Yield:4.05 g (84%) of partly chrystalline XlVa, homogeneous according to TLCin P and C, and [01],, +7.35 (c 1.0; DMF).

XlVb. Cbo Leu Arg (N0 2 NA Starting materials: 1.5 g (3.1 mmoles) of XlVa and 1.43 g (3.7 mmoles) of Cbo Leu OpNP.

Method of synthesis: According to Example lb.

Purification: Gel chromatography on Sephadex Ll-l-20 in MeOH. Yield: 1.6g 86%) of amorphous XlVb, homogeneous according to TLC in P and [01],,9.1 (c 1.0; DMF).

XlVc. Cbo Leu Leu Arg (N0 2 NA Starting materials: 1.35 g (2.26 mmoles)of XlVb and 1.05 g (2.71 mmoles) of Cbo-Leu-OpNP.

Method of synthesis: According to Example lb.

Purification: Gel chromatography on Sephadex Ll-l-20 in MeOH. Yield;1.00 g (62.4%) of partly chrystalline XlV 0, homogeneous according toTLC in P and [01],, 20.6 (c 1.0; DMF).

XlVb. N Bz' Leu Arg (N0 2 NA Starting materials: 990 mg (1.4 mmoles) ofXlVc and 407 mg (1.8 mmoles) of B2 0.

Method of synthesis: According to Example lla.

Purification: Gel chromatography on Sephadex LH-20 in MeOH. Yield: 800mg (84%) of amorphous XlVd, homogeneous according to TLC in? and [01],,13.4 (c 1.0; DMF).

XIV. N Bz Leu Leu Arg 2 -NA HCl Example XV: N Bz Leu Leu Arg l nitro 2NA. I-ICl XV,. Cbo Arg (N 1 nitro 2 NA Starting materials: 3.6 g mmoles)of Cbo Arg (N0 and 2.26 g (12 mmoles) of l nitro 2 naphthylamine.

Method of synthesis: According to Example XIVa.

Purification: Gel chromatography on Sephadex LH-20 in MeOH.

Yield: 3.1 g (58.7%) of amorphous XVa, homogeneous according to TLC in Pand C and [ab 1 1.8 (C 1.0; DMF).

XVb. Cbo Leu Arg (N0 1 nitro 2 NA Starting materials: 950 mg (1.8mmoles) of XVa and 850 mg (2.2 mmoles) of Cbo Leu OpNP.

Method of synthesis: According to Example lb.

Purification: Gel chromatography on Sephadex LI-1-20 in MeOH. Yield: 900mg (78%) of amorphous XVb, homogeneous according to TLC in P and [(11-10.2 (c 1.02; DMF).

XVc. Cbo Leu Leu Arg (N0 l nitro 2 NA Starting materials: 900 mg (1.4mmoles) of XVb and 650 mg (1.7 mmoles) of Cbo Leu OpNP.

Method of synthesis: According to Example lb.

Purification: Gel chromatography on Sephadex LH-20 in MeOH. Yield: 810mg (77%) of amorphous XVc, homogeneous according to TLC in P and [021'-l8.2 (c 1.01: DMF).

XVd. N Bz Leu Leu Arg (N0 1 nitro 2 NA Starting materials: 680 mg (0.88mmole) of XVc and 260 mg (1.15 mmoles) of B2 0.

Method of synthesis: According to Example Ila.

Purification: Gel chromatography on Sephadex LH-20 in MeOH. Yield: 480mg (73%) of amorphous XVd, homogeneous according to TLC in P and [01],,3l.4 (c 0.9; DMF).

XV. N -Bz-Leu-Leu-Arg- 1 -nitro.-2-NA.

HCI

Starting material: 74 mg (0.1 mmole) of XVd.

Method of synthesis: According to Example I.

Purification: Gel chromatography on Sephadex G- in 33%.AcOH. Yield: 47mg (66%) of lyophilized, amorphous XV, chlorine content 4.94%,homogeneous according to TLC in A and [021 33.6 (c 0.72; 50% AcOH).Amino acid analysis showed the following proportions: Leu: 2.0;Arg:'0.9. Y

Example XVI: N Bz Leu Leu Arg 4 nitro 1 NA HCl XVIa. Cbo Arg (N0 4 nitrol NA Starting materials: 3.6 g (10 mmoles) of Cbo Arg (N0 OH and 2.26 g(12 mmoles) of4 nitro l naphthYlamine.

Method of synthesis: According to Example XIVa.

Purification: Gel chromatography on Sephadex LH-20 in MeOH. v Yield: 2.9g (55%) of amorphous XVIa, homogeneous according to TLC in P and C and[01],, l 1.4 (C 1.01; DMF).

XVIb. Cbo Leu Arg (N0 4 nitro l NA Starting materials: 650 mg (1.23mmoles) of XVIa and 560 mg (1.45 mmoles) of Cbo 1 Leu OpNP. Method ofsynthesis: According to Example lb. Purification: Gel chromatography onSephadex LI-I-20 in MeOH. Yield: 520 mg (66%) of amorphous XVIb,homogeneous according to TLC in P and [01] -ll.8 (c 0.2; DMF).

XVIc. Cbo Leu Leu Arg (N02) 4 -'nitro -'1 NA Starting materials: 520 mg(0.81 mmole) of XVIb and 375 mg (0.97 mmole) of Cbo Leu OpNP. Method ofsynthesis: According to Example lb. Purification: Gel chromatography onSephadex@ LH-20 in MeOH. Yield: 378 mg (62%) of partly chrystallineXVIc, homogeneous according to TLC in P and [M 18.0 (c 1.0; DMF).

XVId. N Bz Leu Leu Arg (N0 4 nitro 1 NA Starting materials: 150 mg (0.2mmole) of XVIc and 57 mg (0.25 mmole) of B2 0.

Method of synthesis: According to Example Ila.

Purification: Gel chromatography on Sephadex Ll-I-20 in MeOH. Yield: mg(83%) of amorphous XVId, homogeneous according to TLC in P and [11],30.9 (c 0.95; DMF).

XVLN -Bz-Leu-Leu-Arg-4-nitro-l-NA.

HCI

Starting material: 120 mg (0.165 mmole) of XVId.

Method of synthesis: According to Example I.

Purification: Gel chromatography on Sephadex G-15 in 33% AcOH. Yield: 48mg (42%) of lyophilized, amorphous XVI, chlorine content 4.95%,homogeneous according to TLC in A and [01],, =36.0 (c 0.71; 50% AcOH).Amino acid analysis showed the following proportions Leu: 2.0; Arg: 0.9.

Example XVII: N B2 Leu Leu Lys pNA HCI XVIIa. N BOC Lys (6 Cbo) pNA 6.3g (11.2 mmoles) of N BOC Lys (6 Cbo) OH DCHA are dissolved in 40 ml ofdry, freshly destilled HMPTA at room temperature, where upon 5 g (30.5mmoles) of p-nitro-phenylisocyanate are aded successively with stirringunder completely moisture free conditions. After 24 hours at roomtemperature the reaction mixture is worked up according to thedescription in Example la. The insoluble part, consisting XVllb. N BOCLeu Lys (e Cbo) pNA 2.20 g (4.4 mmoles) of XVlla are dissolved in 10 mlfreshly destilled trifluoroacetic acid under completely moisture freeconditions. The reaction mixture is stirred for one hour at roomtemperature and thereafter it is slowly poured into 150 ml of dryvigerously stirred ether, whereby C1 COOl-l 1-1 Lys (6 Cbo) pNAprecipitates out after cooling. The ether solution is decanted off andthe amorphous residue is treated another three times with 75 ml ofether. After being dried in vacuumover P and NaOH, the yield of thetrifluoroacetic acid salt of the amino acid derivate is quantitative(2.25 g).

2.25 g (4.4 mmoles) of CF;, COOH H Lys (6 Cbo) pNA are dissolved in 10ml DMF. The solution is cooled to 10C and 0.78 ml (5.5 mmoles) Et N isadded to .liberate the amino acid salt. 2.4 g (6.5 mmoles) of BOC LeuOpNP are added and the solution is allowed to reach the roomtemperature. After 3 hours the solution is cooled again to 10C andbuffered with 0.31 ml (2.2 mmoles) of Et N. The buffering procedure isrepeated once more after 2-3 hours. After 24 hours reaction time thesolution is evaporated at 40C in vacuum to dryness. The evaporationresidue is treated three times with ml of destilled water and then driedin vacuum. The crude, dry residue is dissolved in MeOl-l and purified bygel chromatography on a column of Sephadex LH-20 equilibrated with MeOH.

Yield: 1.70 g (70%) of amorphous XVllb, homogeneous according to TLC inP and C, and [01] -4.9 (c 1.1; DMF).

XVlIc. N BOC Leu Leu Lys (e Cbo) pNA Starting materials: 950 mg (1.55mmoles) of XVllb and 1.16 g (3.1 mmoles) of BOC Leu OpNP.

Method of synthesis: According to Example XVllb. Purification: Gelchromatography on Sephadex LH-20 in MeOH.

mg (1.7 mmoles) of B2 0 were added to the solution at 10C, after whichthe procedures of buffering and work up were carried out according toExample XVllb. Purification: Gel chromatography on Sephadex LH-20 inMeOH. Yield: 920 mg (90%) of partly chrystalline XVlld, homogeneousaccording to TLC in P and C, and [11],,

5.95 (c 1.02; DMF).

XVlle. N Bz Leu Leu Lys pNA l-lCl Starting material: 300 mg (0.41 mmole)of XVlld.

Method of synthesis: According to Example 1.

Purification: Gel chromatography on Sephadex G-15 in 33% AcOH. Yield:170 mg (66%) of lyophilized amorphous XVll, chlorine content 5.51%,homogeneous according to TLC in A, and [01],; 50.5 (c 0.62; 50% AcOl-l).Amino acid analysis showed the following proportions: Leu: 2.0; Lys:0.35.

The substrates produced according to the examples were used fordetermination of different enzymes according to the following:

The principle for the determination is based upon the fact that theproduct formed by the enzymatic hydrolysis shows an UV spectrum which isentirely separate from that of the substrate. Thus, the substrateaccording to Example 11 N -Bz-Leu-Leu-Arg-pNA.HCl has an absorptionmaximum at 302 nm with the molar extinction coefficient 12920. Theabsorption of the substrate is insignificant at 405 nm. p-Nitroaniline(pNA), which is formed from the substrate during the enzymatichydrolysis, has an absorption maximum at 380 nm with a molar extinctioncoefficient of 13,200, which at 405 nm has only been reduced to 9620.

Therefore, by means of measuring spectrophotometrically at 405 nm, onecan readily follow the degree of the enzymatic hydrolysis which isproportional to the amount of p-nitro aniline formed. The excess ofsubstrate which is present does not interfere with the measurement atthat wave-length. The circumstances are almost identical for theremaining substrates of the invention, and for this reason thespectrophotometric measurements were throughout made at 405 nm.

The enzymatic reaction can schematically be written in the followingmanner:

E S ES' chr cmophore P E enzyme S substrate Yield: 985 mg (88%) ofamorphous XVllc, homogeneous according to TLC in P and [a] 22.0 (c 1.0;DMF).

XVlId. N Bz Leu Leu Lys (6 Cbo) pNA ES enzyme-substrate complex P and Pproducts k k k and k rate constants 2 Dissociation const. for E5 K(Michaelis const.)

If (S) (E) and k k the following is true:

1( (E 11 X (ES) (1) The rate at which the chromophore P is formed: v k x(ES) k; X (E) X K,..+ s) (2) When all E is bound to S, (ES) (E) and Asevident from equation (2), the constants K,, and

k determine the efficiency of the enzyme substrate for 15 a givenenzyme. For the purpose of determining these constants, the procedure isin principle the following: The enzyme and the substrate are mixed in abuffer solution and the reaction is followed spectrophotometrically for5 min. The concentration of the substrate 20 (S) is varied, while theenzyme concentration (E) for each substrate is kept constant. Theextinction (OD) is plotted as a function of time. From the curveobtained in this manner the tangent difference in extinction per min, AOD/min, from which the amount umol 5 formed p-NA/min (V), may becalculated) at time zero gives the initial reaction rate, v. If 1/v isplotted against 1/(S) K,, and v (of equation (4) are obtained fromthediagram. K and k v ,(E) for trypsin and different enzyme substratesare given in Table 1 and for thrombin in Table 2. The data regarding Kand k is missing in those cases where the values have not beendetermined, or where they could not be calculated because no linearrelation between l/v and 1/(S) was obtained.

A rough evaluation of the enzyme-substrate relationship for differentsubstrates: may be obtained by means of comparing the amount ofp-nitro-aniline formed per min. and per m1 at thesame concentration ofthe substrates. This is shown in Table 3 for trypsin, in Table 4 forthrombin and in Table 5 for plasmin.

The letters NIH used in the Tables refer to units of the NationalInstitute of Health. The trypsin unit is equal to the hydrolysis of onepmole of N -tosylarginine-methyl ester hydrochloride (TAME) per minuteat 25C and pH 8.1 in the presence of 0.01 M calcium ion, and the trypsinwas obtained from Worthington Biochemical Corporation, Freehold, N.J.,USA. The plasmin was obtained from AB Kabi, Stockholm, Sweden, and 1 mgwas equal to 3 casein units (CU).

Table 1 Trypsin-activity, K,, and k k X 10 v Substrate Enzyme K, X 10"Substrate conc. conc. I

(umole/l) (NIH/ml) (mole/l) (umole/min, NIH/ml BAPNA 250-666 75 16 8,3 l33.6l12. 0 6 7.40 330 ll 35.0150.0 3.3 0.64 276 IV 35.0150.0 3.3 0.17 71Table 2 Thrombin-activity, K and lo,

' Substrate Enzyme l(,,, X 10" k X 10" Substrate conc. cone.

(umole/l) (NIH/m1) (mole/l) (umole/min, NIH/m1) BAPNA 250-666 50 -6 -1 lI 33.6-1 12.0 10 2.92 40 Table 3 Trypsin-activity Substrate Substratecone. Enxyme conc. Formed p-nitroaniline nmoles per ml units per mlnmoles per min. per ml BAPNA 333.0 50.0 1.04 11 66.9 0.0333 14.2 11166.7 16.8 [V 67.0 10.2 V 66.3 22.7 VI 66.8 12.8 Vll 66.2 16.6 Vlll 66.717.1 1X 67.0 12.0 X 67.1 15.5 XI 66.3 12.4 Xll 65.9 16.5 Xlll 66.9 g16.7

Table 4 Thrombin-activity Plasmin-activity Substrate Substrate conc.Enzyme conc. Formed p-nitroaniline nmoles per ml CU-units per ml nmolesper min. per ml II 66.9 0.167 2.7 III 66.7 17.2 IV 67.0 2.7 V 66.5 7.7VI 668 2.8 VII 66.2 2.2 VIII 66.7 4.0 IX 67.0 1.5 X 67.1 17.2 XI 66,32.0 XII 65.9 1.5 XIII 66.9 17.9 21.3

XVII 67.0

The Tables 1 5 clearly demonstrate the advantages carbon atoms; X isselected from the group consisting of the enzyme substrates of theinvention when comof methylene and a single bond; R is selected from thepared with the amide substrate previously used for group consisting ofstraight, branched and cyclic alkyl yp y ns of their greater sensitivitygroups having 3-8 carbon atoms; R, is selected from the new substratesmake the determination of small the group consisting of straight,branched and cyclic amounts of enzyme possible without jeopardizing thealkyl groups having 3-8 carbon atoms, phenyl and benexactness of thedetermination. This is very important zyl; n is 3 or 4; R is selectedfrom the group consisting from a clinical point of view, since itsimplifies the colof hydrogen and guanyl; and R is selected from thelection of specimen. group consisting of phenyl, nitrophenyl, methylni-What is claimed is: trophenyl, naphthyl, nitronaphthyl, quinolyl, andnil. Substrate with a high susceptibility to peptide peptroquinolyl.

tidohydrolases which is represented by the formula: 2. The substrate ofclaim 1 wherein R is benzoyl.

o o 0 1| 1| ll R1 N x CH c NH CH c NH CH c NH R6 or acid addition saltsthereof, where R is selected from 3. The substrate of claim 1 wherein Ris hydrogen. the group consisting of hydrogen, an acyl alkyl- 4. Thesubstrate of claim 1 wherein R is hydrogen. carbonyl having 1-12 carbonatoms, a w-aminoalkyl- 5. The substrate of claim 1 wherein n is 3 and Ris carbonyl having I-l2 carbon atoms in a straight chain, guanyl.cyclohexyl-carbonyl, a m-cyclohexylalkyl-carbonyl 6. The substrate ofclaim 1 wherein n is 4 and R is having l-6 carbon atoms in a straightchain, 4- hydrog naminomethyI-cyclohexylcarbonyl, benzoyl, a 5 7. Thesubstrate of claim 1 wherein R is nitrophenyl. w-phenylalkyl-carbonylhaving l-6 carbon atoms in a 8. The substrate of claim 1 wherein R isnaphthyl. straight chain, benzenesulphonyl and 4-toluene- 9. Thesubstrate of claim 1 wherein R is nitronaphtsulphonyl; R is selectedfrom the group consisting of hyl. hydrogen, phenyl, cyclohexyl and analkyl having l-6 10. The substrate of claim 1 wherein said substrate is.2 1 -benzoyl-leucyl-leucyl-arginine-p-nitroanilide or the l-lCl saltthereof.

11. The substrate of claim 1 wherein said substrate is N-benzoyl-N-cyclohexyl-B-alanyl-valyl-arginine-pnitroanilide or the HClsalt thereof.

12. The substrate of claim 1 wherein said substrate is N"-benzoyl-leucyl-valyl-arginine-p-nitroanilide or the HCl salt thereof.

13. The substrate of claim 1 wherein said substrate is N-benzoyl-isoleucyl-valyl-arginine-p-nitroanilide or the HCl saltthereof.

14. The substrate of claim 1 wherein said substrate is N-benzoyl-valyl-isoleucyl-arginine-p-nitroanilide or the HCl saltthereof. 15. The substrate of claim 1 wherein said substrate is

1. SUBSTRATE WITH A HIGH SUSCEPTIBILITY TO PEPTIDE PEPTIDOHYDROLASESWHICH IS REPRESENTED BY THE FORMULA:
 2. The substrate of claim 1 whereinR1 is benzoyl.
 3. The substrate of claim 1 wherein R1 is hydrogen. 4.The substrate of claim 1 wherein R2 is hydrogen.
 5. The substrate ofclaim 1 wherein n is 3 and R5 is guanyl.
 6. The substrate of claim 1wherein n is 4 and R5 is hydrogen.
 7. The substrate of claim 1 whereinR6 is nitrophenyl.
 8. The substrate of claim 1 wherein R6 is naphthyl.9. The substrate of claim 1 wherein R6 is nitronaphthyl.
 10. Thesubstrate of claim 1 wherein said substrate is N-benzoyl-leucyl-leucyl-arginine-p-nitroanilide or the HCl salt thereof.11. The substrate of claim 1 wherein said substrate is N-benzoyl-N-cyclohexyl- Beta -alanyl-valyl-arginine-p-nitroanilide or theHCl salt thereof.
 12. The substrate of claim 1 wherein said substrate isN -benzoyl-leucyl-valyl-arginine-p-nitroanilide or the HCl salt thereof.13. The substrate of claim 1 wherein said substrate is N-benzoyl-isoleucyl-valyl-arginine-p-nitroanilide or the HCl saltthereof.
 14. The substrate of claim 1 wherein said substrate is N-benzoyl-valyl-isoleucyl-arginine-p-nitroanilide or the HCl saltthereof.
 15. The substrate of claim 1 wherein said substrate is N-benzoyl-valyl-leucyl-arginine-p-nitroanilide or the HCl salt thereof.16. The substrate of claim 1 wherein said substrate is N-benzoyl-isoleucyl-leucyl-arginine-p-nitroanilide or the HCl saltthereof.
 17. The substrate of claim 1 wherein said substrate is N-benzoyl-leucyl-leucyl-arginine-2-naphthylamide or the HCl salt thereof.18. The substrate of claim 1 wherein said substrate is N-benzoyl-leucyl-leucyl-arginine-4-nitro-1-naphthylamide or the HCl saltthereof.
 19. The substrate of claim 1 wherein said substrate is N-benzoyl-leucyl-leucyl-lysine-p-nitroanilide or the HCl salt thereof.